UDP-glucuronyltransferase, a hepatic microsomal enzyme, metabolizes bilirubin, hormones and drugs. It has been suggested that this enzyme is heterogeneous. The purpose of this research is to determine the heterogeneity of rat hepatic UDP-glucuronyltransferase. Using as a starting point, a method developed by the principal investigator, solubilized UDP-glucuronyltransferase will be further purified with the aim of physically separating activities toward the following substrates: o-aminophenol, p-nitrophenol, bilirubin, morphine and aniline. Methods to be employed include Sephadex chromatography, sucrose density gradient centrifugation and affinity chromatography. Characterization of the enzyme and enzyme kinetics will be determined in purified preparations to determine if differentiation of UDP-glucuronyltransferase can be made on the basis of its catalytic behavior. Further evidence for heterogeneity will be obtained by determining the kinetic constants of purified UDP-glucuronyltransferase obtained from 3-methylcholanthrene- induced rats and comparing them with controls. Particular attention will be paid to the Michaelis constants of UDP-glucuronic acid, the common substrate, which will be determined using each of the aforementioned acceptor substrates. Finally, the development of UDP- glucuronyltransferase will be determined in rats of various ages. That is, the sequence of appearance of activity toward each of the substrates will be assayed with the aim of ascertaining whether or not there is a non-synchronous appearance of activity toward the different substrates. Purified enzyme will be used.